An emerging approach to the investigation of disease and cellular state involves the analysis of the complete set of RNA molecules—the “transcriptome”—expressed by a cell or tissue sample (see, Devaux, F. (2001) “TRANSCRIPTOMES, TRANSCRIPTION ACTIVATORS AND MICROARRAYS,” FEBS Lett. 498:140-4; see also, U.S. Pat. Nos. 6,221,600; 6,303,308; and 6,461,814; European Patent Publications Nos. EP 0970202a2; 1174521a3; and 1190044a2; Japanese Patent Application No. JP 2002142765a2, and PCT International Patent Publications Nos. WO 0077214a3; WO 0138577a3; WO 02068466a2; WO 0210449a2; WO 0246465a2 and WO 9832847a2).
High-throughput gene expression array and proteomic technologies make possible the simultaneous analysis of thousands of mRNA transcripts and proteins, allowing a global view of the molecular events associated with normal cellular processes and disease states (Schena, M. et al. (1995) “QUANTITATIVE MONITORING OF GENE EXPRESSION PATTERNS WITH A COMPLEMENTARY DNA MICROARRAY,” Science 270: 467469; Schena, M. et al. (1998) “MICROARRAYS: BIOTECHNOLOGY'S DISCOVERY PLATFORM FOR FUNCTIONAL GENOMICS,” Trends Biotechnol. 16: 301-306; DeRisi, J. et al. (1996) “USE OF A cDNA MICROARRAY TO ANALYSE GENE EXPRESSION PATTERNS IN HUMAN CANCER,” Nat. Genet. 14: 457-460; Chee, M. et al. (1996) “ACCESSING GENETIC INFORMATION WITH HIGH-DENSITY DNA ARRAYS. Science 274: 610-614; Lander, E. (1999) “Array Of Hope.” Nat. Genet. 21: 3-4; Emmert-Buck, M. R. et al. (2000) “A STRATEGIC APPROACH FOR PROTEOMIC ANALYSIS OF HUMAN TUMORS,” Mol. Carcin. 27: 1-8; Emmert-Buck, M. R. et al. (2000) “MOLECULAR PROFILING OF CLINICAL TISSUE SPECIMENS: FEASIBILITY AND APPLICATIONS,” Am. J Pathol. 156: 1109-1115; Celis, J. et al. (2000) “GENE EXPRESSION PROFILING: MONITORING TRANSCRIPTION AND TRANSLATION PRODUCTS USING DNA MICRO ARRAYS AND PROTEOMICS,” FEBS Lett. 480: 2-16; Anderson, N. L. et al. (1998) “PROTEOME AND POTEOMICS: NEW TECHNOLOGIES, NEW CONCEPTS, AND NEW WORDS,” Electrophoresis 19: 1853-1861; Duggan, D. J. et al. (1999) “EXPRESSION PROFILING USING CDNA MICROARRAYS,” Nat. Genet. 21: 10-14; Khan, J. et al. (1999) “EXPRESSION PROFILING IN CANCER USING CDNA MICROARRAYS,” Electrophoresis 20: 223-229; Lipshutz, R. J. et al. (1999) “HIGH DENSITY SYNTHETIC OLIGONUCLEOTIDE ARRAYS,” Nat. Genet. 21: 20-24; Lockhart, D. I. et al. (1996) “EXPRESSION MONITORING BY HYBRIDIZATION TO HIGH-DENSITY OLIGONUCLEOTIDE ARRAYS,” Nat. Biotechnol. 14: 1675-1680; Velculescu, V. et al. (1995) “SERIAL ANALYSIS OF GENE EXPRESSION,” Science 270: 484-487; Liotta, L. et al. (2000) “MOLECULAR PROFILING OF HUMAN CANCER,” Nature Reviews Genetics 1: 48-56).
The emergence of transcriptome analysis has, however, been encumbered by the limitations of existing methodologies. Typically, such technologies identify a subset of genes (from a few dozen to several hundred) whose expression profile provides novel insight into cellular physiology and/or allows disease states to be segregated on a molecular rather than a phenotypic basis (Perou, C. et al. (2000) “MOLECULAR PORTRAITS OF HUMAN BREAST TUMOURS,” Nature 406: 747-752; Alizadeh, A. A. et al. (2000) “DISTINCT TYPES OF DIFFUSE LARGE B-CELL LYMPHOMA IDENTIFIED BY GENE EXPRESSION PROFILING,” Nature 403: 503-511; Dhanasekaran, S. et al. (2001) “DELINEATION OF PROGNOSTIC BIOMARKERS IN PROSTATE CANCER,” Nature 412: 822-826; Hedenfalk, I. et al. (2001) “GENE-EXPRESSION PROFILES IN HEREDITARY BREAST CANCER,” N Engl. J. Med. 344: 539-548; Golub, T. R. et al. (1999) “MOLECULAR CLASSIFICATION OF CANCER: CLASS DISCOVERY AND CLASS PREDICTION BY GENE EXPRESSION MONITORING. Science 286:531-537; Klose, J. (1999) “GENOTYPES AND PHENOTYPES,” Electrophoresis 20: 643-652; Strausberg, R. L. et al. (2000) “THE CANCER GENOME ANATOMY PROJECT: BUILDING AN ANNOTATED GENE INDEX,” Trends Genet. 16:103-106; Zhang, L. et al. (1997) “GENE EXPRESSION PROFILES IN NORMAL AND CANCER CELLS,” Science 276: 1268-1272). Although these studies provide valuable information, it is desirable to independently confirm and quantitatively measure the expression level of each of the genes of interest. Prior to the advent of the present invention, this represented a significant challenge in terms of time and effort. Moreover, the amount of biological sample available for subsequent investigation is often limiting, particularly in the case of developmental biology samples and clinical specimens.
Although transcriptome analysis can be conducted by performing multiple Northern blots (Aldaz, C. M. et al., (2002) “SERIAL ANALYSIS OF GENE EXPRESSION IN NORMAL P53 NULL MAMMARY EPITHELIUM,” Oncogene 21:6366-6376), this approach can be laborious and time-consuming (see, Su, A. I. et al. (2002) “LARGE-SCALE ANALYSIS OF THE HUMAN AND MOUSE TRANSCRIPTOMES,” Proc. Natl. Acad. Sci USA 99:4465-70). More fundamentally, such an analysis is inherently biased against low abundance transcripts.
Various protocols have likewise been developed to generate cDNA libraries from globally amplified RNA of single cells (Belyaysky, A et al. (1989) “PCR-BASED CDNA LIBRARY CONSTRUCTION: GENERAL CDNA LIBRARIES AT THE LEVEL OF A FEW CELLS,” Nucl. Acids Res. 17:2919-2932; Brady, G. et al. (1993) “CONSTRUCTION OF CDNA LIBRARIES FROM SINGLE CELLS,” Meth. Enzymol. 225:611-623; Karrer, E. E. et al. (1995) “IN SITU ISOLATION OF MRNA FROM INDIVIDUAL PLANT CELLS: CREATION OF CELL-SPECIFIC CDNA LIBRARIES,” Proc. Natl. Acad. Sci. USA 92:3814-3818), and cDNA microarrays have been used to analyze gene expression patterns (DeRisi, J. et al. (1996) “USE OF A CDNA MICROARRAY TO ANALYSE GENE EXPRESSION PATTERNS IN HUMAN CANCER” Nature Genetics 14:457-60; Li, S. et al. (2001) “COMPARATIVE GENOME-SCALE ANALYSIS OF GENE EXPRESSION PROFILES IN T CELL LYMPHOMA CELLS DURING MALIGNANT PROGRESSION USING A COMPLEMENTARY DNA MICROARRAY,” Amer. J. Pathol. 158:1231-1237; Saha, S. et al. (2002) “USING THE TRANSCRIPTOME TO ANNOTATE THE GENOME,” Nat. Biotechnol. 20:508-512; Bono, H. et al. (2002) “FUNCTIONAL TRANSCRIPTOMES: COMPARATIVE ANALYSIS OF BIOLOGICAL PATHWAYS AND PROCESSES IN EUKARYOTES TO INFER GENETIC NETWORKS AMONG TRANSCRIPTS,” Curr Opin Struct Biol. 12:355-361; Schena, M. et al. (1995) “QUANTITATIVE MONITORING OF GENE EXPRESSION PATTERNS WITH A COMPLEMENTARY DNA MICROARRAY” Science 270:467-70; Anisimov, S. et al. (2002) “A QUANTITATIVE AND VALIDATED SAGE TRANSCRIPTOME REFERENCE FOR ADULT MOUSE HEART,” Genomics 80:213-222).
Unfortunately, however, all such protocols have drawbacks, including the selective amplification of the 3′ ends of a transcript, insufficient sensitivity in amplification (Klein, C. A. et al. (2002) “COMBINED TRANSCRIPTOME AND GENOME ANALYSIS OF SINGLE MICROMETASTATIC CELLS,” Nat. Biotechnol. 20:387-92) and the problem of distinguishing critical transcript species from merely abundant transcripts (Curtis, R. K. et al. (2002) “CONTROL ANALYSIS OF DNA MICROARRAY EXPRESSION DATA,” Mol. Biol. Rep. 29:67-71). While microarrays permit one to compare transcriptomes of different cells and tissues, they do not retain information concerning the architecture or location of the detected transcripts. Techniques of in situ hybridization and amplification have been developed to permit the localization of RNA transcripts, however such techniques focus on one or a small number of genes and do not assess the expression of the transcriptome (see, e.g., Nuovo, G. J. (2001) “CO-LABELING USING IN SITU PCR: A REVIEW,” J. Histochem. Cytochem. 49:1329-1339; Moore, J. G. et al. (2000) “HER-2/NEU GENE AMPLIFICATION IN BREAST IMPRINT CYTOLOGY ANALYZED BY FLUORESCENCE IN SITU HYBRIDIZATION: DIRECT COMPARISON WITH COMPANION TISSUE SECTIONS,” Diagn. Cytopathol. 23:299-302; Seeds, M. C. et al. (2000) “CELL-SPECIFIC EXPRESSION OF GROUP X AND GROUP V SECRETORY PHOSPHOLIPASES A(2) IN HUMAN LUNG AIRWAY EPITHELIAL CELLS,” Amer. J. Respir. Cell. Mol. Biol. 23:37-44).
Various approaches have been attempted to “capture” the 2-dimensional positional relationship between molecules of a sampled array. A paraffin block has been described (website with the host name of “cmag”, domain name of “cit.nih.gov” and file extension “Tissuearray.htm) in which multiple cores (50-500) of tissue are placed in an organized grid. The device is a said to be amenable for use in a variety of experiments, including immunohistochemistry, immunofluorescence, FISH, in situ hybridization, and to provide a high throughput platform for tissue, in which hundreds of samples can be analyzed at one time, and multiple experiments can be performed on the same array (see, the website with the host name “www”, domain name of “laborel.no”, and file extension “Acrobat/Biogenex/Biolink%20VOL.pdf). Microwell and microtiter plates (e.g., Thermo Labsystems 384-Well Solid Microtiter Plate) are example of 2-dimensional arrays of partitioned grids or chambers.
The problem of detecting high relevance, low abundance, transcripts is of particular significance in the analysis of complex tissue samples. Advanced technologies, such as the “Gene Chip” are reportedly able to detect no more than 30% of the transcripts present in complex tissue samples (Evans, S. J. et al. (2002) “EVALUATION OF AFFYMETRIX GENE CHIP SENSITIVITY IN RAT HIPPOCAMPAL TISSUE USING SAGE ANALYSIS. SERIAL ANALYSIS OF GENE EXPRESSION,” Eur. J. Neurosci. 16:409-13; Piper, M. D. W. et al. (2002) “REPRODUCIBILITY OF OLIGONUCLEOTIDE MICROARRAY TRANSCRIPTOME ANALYSES: AN INTERLABORATORY COMPARISON USING CHEMOSTAT CULTURES OF SACCHAROMYCES CEREVISIAE,” J. Biol. Chem. 277:37001-37008). Where relevant cells (i.e., those associated with the production of high relevance, low abundance, transcripts) can be identified, techniques such as microdissection or laser-capture microscopy may be employed (Emmert-Buck, M. R. et al. (1996) “LASER CAPTURE MICRODISSECTION,” Science 274:998-1001; Bonner, R. F. et al. (1997) “LASER CAPTURE MICRODISSECTION: MOLECULAR ANALYSIS OF TISSUE,” Science 278:1481-1483), however, in many cases such relevant cells have not been identified, or cannot be detected.
In particular, there is an important need for new technologies that facilitate follow-up analysis of array- and proteomic-derived data. Although many such approaches are under development each has its particular weaknesses (see, for example, Kononen, J. et al. (1998) “TISSUE MICRO ARRAYS FOR HIGH-THROUGHPUT MOLECULAR PROFILING OF TUMOR SPECIMENS,” Nat. Med. 4: 844-847; Berndt, P. et al. (1999) “RELIABLE AUTOMATIC PROTEIN IDENTIFICATION FROM MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRIC PEPTIDE FINGERPRINTS,” Electrophoresis 20: 3521-3526; Binz, P. A. et al. (1999) “A MOLECULAR SCANNER TO AUTOMATE PROTEOMIC RESEARCH AND TO DISPLAY PROTEOME IMAGES,” Anal. Chem. 71: 4981-4988; Bubendorf, L. et al. (1999) “SURVEY OF GENE AMPLIFICATIONS DURING PROSTATE CANCER PROGRESSION BY HIGH-THROUGHOUT FLUORESCENCE IN SITU HYBRIDIZATION ON TISSUE MICROARRAYS,” Cancer Res. 59: 803-806; Celis, J. E. et al. (1999) “2D PROTEIN ELECTROPHORESIS: CAN IT BE PERFECTED?” Curr. Opin. Biotechnol. 10:16-21; Humphery-Smith, I. (1998) “PROTEOMICS: FROM SMALL GENES TO HIGH-THROUGHPUT ROBOTICS,” J. Protein Chem. 17:524-525; Herbert, B. (1999) “ADVANCES IN PROTEIN SOLUBILIZATION FOR TWO-DIMENSIONAL ELECTROPHORESIS,” Electrophoresis 20: 660-663; Liu. Y. et al. (1999) “ACTIVITY-BASED PROTEIN PROFILING: THE SERINE HYDROLASES,” Proc. Natl. Acad. Sci. USA. 96: 14694-14699; Lueking, A. et al. (1999) “PROTEIN MICRO ARRAYS FOR GENE EXPRESSION AND ANTIBODY SCREENING,” Anal. Biochem. 270:103-111; Quadroni, M. et al. (1999) “PROTEOMICS AND AUTOMATION,” Electrophoresis 20: 664-677; Yates, J. R., 3rd (2000) “MASS SPECTROMETRY: FROM GENOMICS TO PROTEOMICS,” Trends Genet. 16: 5-8; Sidransky, D. (2000) “EMERGING MOLECULAR MARKERS OF CANCER,” Nature Reviews Cancer 2: 210-219).
Thus, despite all such advances, the development of a global amplification system remains “the most critical hurdle” to transcriptome analysis. (Klein, C. A. et al. (2002) “COMBINED TRANSCRIPTOME AND GENOME ANALYSIS OF SINGLE MICROMETASTATIC CELLS,” Nat. Biotechnol. 20:387-92). A need thus remains for an apparatus and method that would permit multiple, preferably simultaneous, manipulations of the biomolecules present in a two-dimensional array, such as a gel, or other solid support. The present invention is directed to this and other needs.